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CARDIOVASCULAR

Raloxifene Relaxes Rat Pulmonary Arteries and Veins: Roles of Gender, Endothelium, and Antagonism of Ca²⁺ Influx

Department of Physiology, Chinese University of Hong Kong, Hong Kong, China (Y.-C.C., F.-P.L., X.Y., C.-W.L., Y.H.); and Department of Pharmacology, University of Hong Kong, Hong Kong, China (P.M.V.)

Received September 16, 2004; accepted November 12, 2004.

	Abstract

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Effects of raloxifene have been documented in the systemic circulation. However, its impact on the pulmonary circulation is unclear. The present study investigated the role of gender, endothelial modulation, and Ca²⁺ channel in relaxations evoked by raloxifene in rat pulmonary arteries and veins. Vascular responses were studied on isolated pulmonary blood vessels mounted in a myograph and constricted by U46619 [GenBank] (9,11-dideoxy-11,9-epoxymethanoprostaglandin F₂). Constrictions to CaCl₂ were studied in Ca²⁺-free, 60 mM K⁺ solution. Changes in the intracellular calcium ion concentration ([Ca²⁺]_i) in vascular smooth muscle were measured using a calcium fluorescence imaging method. Raloxifene was more effective in relaxing U46619 [GenBank] -constricted pulmonary arteries from male than female rats. Raloxifene-induced relaxation was unaffected by ICI 182,780 [7-[9-[(4,4,5,5,5,-pentafluoropentyl)-sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17-diol], inhibition of the nitric oxide (NO) pathway, or removal of the endothelium. In arteries without endothelium, raloxifene attenuated CaCl₂-induced constriction and CaCl₂-stimulated increase in [Ca²⁺]_i with similar potencies. Raloxifene caused endothelium-independent relaxations in pulmonary veins, albeit to a lesser degree than in pulmonary arteries. The venous responses showed a gender difference because raloxifene was more potent in male veins. In summary, raloxifene relaxed rat pulmonary arteries, and this effect did not involve the endothelium/NO or ICI 182,780-sensitive estrogen receptors. Raloxifene, like nifedipine, reduced constriction and [Ca²⁺]_i increase in response to CaCl₂ in high K⁺ solution. Raloxifene also relaxed high K⁺-constricted pulmonary veins. Our data indicate that raloxifene acutely relaxes rat pulmonary blood vessels primarily via inhibition of Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Finally, raloxifene induced more relaxation in blood vessels isolated from male than female rats.

Female rats exposed to chronic hypoxia exhibited less pulmonary arterial hypertension (Rabinovitch et al., 1981) and right ventricular hypertrophy (McMurtry et al., 1973) compared with age-matched male rats. Similar to the systemic circulation, one mechanism for estrogen modulation of pulmonary artery relaxation may be mediated by augmented nitric oxide (NO) function (Gonzales et al., 2001).

Many studies have explored the mechanisms for SERM modulation of vascular tone in systemic arteries and veins (Figtree et al., 1999; Bracamonte et al., 2002; Tsang et al., 2004a). Vasorelaxation to raloxifene in females is influenced by ovarian hormonal status (Bracamonte et al., 2002). Two main mechanisms reported for the vascular action of SERMs in systemic vascular tissues are up-regulation of endothelial NO production (Figtree et al., 1999; Bracamonte et al., 2002) and inhibition of L-type voltage-sensitive Ca²⁺ channels (Tsang et al., 2004b). However, no studies have examined SERM regulation of vasomotor activity in the pulmonary vascular circulation and its potential as a new drug in the treatment of pulmonary arterial hypertension. Therefore, we investigated the vascular effects of raloxifene, the roles of endothelial modulation, estrogen receptors, and Ca²⁺ channel antagonism in isolated rat pulmonary arteries and veins and gender differences in the action of raloxifene.

	Methods and Materials

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Blood Vessel Preparation. This study was approved by the Animal Research Ethics Committee of Chinese University of Hong Kong. This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH Publication No. 85-23, revised 1996). Female and male Sprague-Dawley rats weighing 250 to 300 g were euthanized. The lungs were carefully dissected out and placed in ice-cold Krebs' solution. Second-order intralobar pulmonary arteries (internal diameter, 200 µm) and main pulmonary veins (internal diameter, 490 µm) were dissected free from lungs, and the surrounding connective tissue was cleaned off under a dissecting microscope. Each blood vessel was cut into two 1-mm ring segments. The individual ring was mounted between two tungsten wires (40 µm in diameter) for the measurement of isometric tension in a 5-ml organ chamber filled with Krebs' solution. Each wire was fixed to the mounting jaws of the myograph (Danish Myo Technology A/S, Aarhus, Denmark). The chamber solution was continuously bubbled with 95% O₂/5% CO₂ at 37°C (pH 7.4). All the rings were placed under an optimal resting tension (1 mN), which was the minimum level of stretch giving the largest force development in 60 mM K⁺ solution, as determined by the length-tension relationship (Nyhan et al., 2002). In most experiments, the endothelial layer was removed mechanically by gently rubbing the luminal surface with a tungsten wire, and the functional removal was verified by the lack of relaxation to 3 µM acetylcholine.

Protocols. After mounting (30 min), rings were first constricted by 100 nM U46619 [GenBank] and subsequently challenged with acetylcholine to confirm the integrity or removal of the endothelium. Then they were washed in Krebs' solution to restore tension to baseline level and allowed to stabilize for 60 min. The role of endothelium/NO and estrogen receptors was first examined in pulmonary relaxant responses to raloxifene. For this series of experiments, rings were exposed for 30 min to each inhibitor (100 µM N^G-nitro-L-arginine methyl ester, 3 µM1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one, or 10 µM ICI 182,780) before the addition of U46619 [GenBank] . Once steady blood vessel tone was obtained, raloxifene was applied cumulatively to the bathing solution. Only one concentration-response curve to raloxifene was obtained per ring in the presence of each inhibitor. The ability of raloxifene to modulate Ca²⁺ influx via L-type voltage-sensitive Ca²⁺ channels was studied on constrictions to CaCl₂ in rings without endothelium. For this set of experiments, two consecutive concentration-dependent constrictions to CaCl₂ were obtained in control and in the presence of raloxifene (0.1-10 µM, 30-min incubation) before repeating the second concentration-response curve. For constructing CaCl₂ concentration-response curve, arterial rings were rinsed three times in a Ca²⁺-free solution containing 30 µM Na₂-EGTA and then incubated in Ca²⁺-free, 60 mM K⁺ solution before the cumulative addition of CaCl₂ (0.1-5 mM). The effect of 1 µM nifedipine was tested as control. The relaxing effect of raloxifene also was studied on 60 mM K⁺-constricted rings in normal Ca²⁺-containing Krebs' solution. We finally examined the effect of raloxifene on isolated rat pulmonary veins and the role of the endothelium/NO.

Measurement of Vascular Smooth Muscle [Ca²⁺]_i. [Ca²⁺]_i was measured in Fura-2-loaded pulmonary artery smooth muscle using the ratio imaging. Rings were incubated for 1 h at 22°C in a Fura-2 loading solution that contained 10 µM Fura-2 AM and 0.025% pluronic F-127 (to prevent Fura-2 secretion). Thereafter, extracellular Fura-2 AM was removed by repetitive washing in Krebs' solution. Rings then were perfused for 20 min with Krebs' solution at 2 ml/min (37°C) to permit cleavage of intracellular Fura-2 AM into active Fura-2 by esterases. Because of the Fura-2 photosensitivity, precautions were taken to avoid extensive photobleaching. A shutter blocked the excitation light when no fluorescence measurement was performed.

The [Ca²⁺]_i imaging setup was modified from that described by Huang et al. (2000). In brief, after Fura-2 loading, cut-open artery rings without endothelium were mounted onto a block of silicone elastomer with stainless steel wires and pins (Sylgard; World Precision Instruments, Inc, Sarasota, FL), which was then fixed into a base plate of a custom-made flow chamber. The base plate was covered with a gasket and cover glass (24 x 32 mm; thickness, no. 1; Menzel-Glaser, Braunschweig, Germany) and affixed by screws. There was a 1-mm gap between the vessel and the cover glass to allow flow passage. This arrangement also allows free vessel movement in response to the addition of vasoactive drugs. After mounting, the flow chamber was placed on an inverted microscope and perfused at 2 ml/min with Krebs' solution at 37°C using a six-channel perfusion pump (Watson Marlow Bredel Pumps Inc., Wilmington, MA).

Fura-2-loaded arterial tissues were viewed through a Nikon CF Fluor 20x objective on an inverted Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan). Fura-2 was excited using a collimated beam of light from a 75-W xenon arc lamp that passed through a D-104 microscope photometer (Photon Technology International, Mon-mouth Junction, NJ) that altered wavelengths from 340 to 380 nm using an OC-4000 optical chopper (Photon Technology International). A photomultiplier tube collected the emitted light at 510 nm. Data acquisition and analysis were performed using FELIX 1.21 software (Photon Technology International).

After mounting, the arterial tissues were allowed to recover for 30 min at 37°C and then exposed for 30 min to a Ca²⁺-free, 60 mM K⁺ perfusion solution. They subsequently were perfused with 60 mM K⁺ containing CaCl₂ (0.1-3 mM) to construct the first concentration-response curve. Rings were rinsed first in Ca²⁺-free solution and then in Ca²⁺-free, 60 mM K⁺ solution to allow returning of the Ca²⁺ level to baseline and finally incubated for 30 min with 300 nM or 10 µM raloxifene before repeating the second CaCl₂ concentration-response curve.

Drugs. Acetylcholine [2-(acetyloxy)-N,N,N-trimethylethanaminium], U46619 [GenBank] , L-NAME, ODQ, and nifedipine [1,4-dihydro-2,6-dimethyl-4(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester] were purchased from Sigma-Aldrich (St. Louis, MO). ICI 182,780 was purchased from Tocris Cookson Inc. (Ellisville, MO). Raloxifene [[6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophen-3-yl][4-[2-piperidino-ethoxy]phenyl]ketone hydrochloride] was a gift from Eli Lilly & Co. (Indianapolis, IN). U46619 [GenBank] , raloxifene, and nifedipine were dissolved in dimethyl sulfoxide; others were dissolved in distilled water. Further dilution was made from stock solutions. Krebs' solution contained 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, and 11 mM D-glucose. High K⁺ solution was prepared by replacing Na⁺ with an equimolar amount of K⁺ to retain constant ionic strength.

Data Analysis. Results are the mean ± S.E.M. of rings from n rats. Increases in constrictive force were expressed as percentages of the maximal response obtained in the first concentration-dependent constriction to CaCl₂. Concentration-response curves were constructed based on responses to cumulative drug concentrations and analyzed by nonlinear curve fitting using GraphPad software version 3.0 (GraphPad Software, Inc., San Diego, CA). The negative logarithm of the dilator (or constrictor) concentration that caused half (pD₂ or pEC₅₀) of the maximal response (E_max) was obtained. For statistical analysis, two-tailed Student's t test or one-way analysis of variance (ANOVA) followed by Newman-Keuls test was used when more than two groups were compared. Individual concentration-response curves also were compared using a two-way ANOVA followed by a post hoc test. P < 0.05 was considered to be significant.

	Results

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Effects of Raloxifene on Pulmonary Arteries. U46619 [GenBank] constricted rat pulmonary arteries to a comparable degree in both genders (tension: 3.71 ± 0.60 mN with endothelium and 3.73 ± 0.36 mN without endothelium in female, P > 0.05; 3.13 ± 0.51 mN with endothelium and 2.98 ± 0.34 mN without endothelium in male, P > 0.05). On U46619 [GenBank] preconstriction, raloxifene caused relaxations of arteries with and without endothelium (pD₂: 4.78 ± 0.13 versus 5.13 ± 0.13, P > 0.05 in female, Fig. 1A; 5.34 ± 0.15 versus 5.53 ± 0.08, P > 0.05 in male, Fig. 1D). Neither L-NAME (NO synthase inhibitor) nor ODQ (guanylate cyclase inhibitor) affected relaxation to raloxifene in female (Fig. 1B) and male (Fig. 1E) arteries. Likewise, ICI 182,780 was without effect on arteries from both genders (Fig. 1, C and F). ICI 182,780 may act as a partial estrogen receptor agonist (Bracamonte et al., 2002), but this agent (0.1-50 µM) did not affect U46619 [GenBank] -induced constriction (data not shown). Vehicle (dimethyl sulfoxide) did not influence U46619 [GenBank] -induced tone in arteries with and without endothelium (Fig. 1, A and D).

View larger version (37K): [in this window] [in a new window]   Fig. 1. Concentration-dependent relaxations to raloxifene in (A) female and (D) male rat pulmonary arteries with and without endothelium, in arteries with endothelium treated by 100 µM L-NAME or 3 µM ODQ (B and E), or by 10 µM ICI 182,780 (C and F). Arteries were constricted by U46619 [GenBank] after a 30-min incubation with each inhibitor. Results are mean ± S.E.M. of 7 or 10 experiments.

There was a gender difference in relaxations to raloxifene in U46619 [GenBank] -constricted arteries (pD₂: 4.78 ± 0.13 in female and 5.34 ± 0.15 in male, P < 0.05; Fig. 2A), but this difference was absent in 60 mM K⁺-constricted arteries (pD₂: 5.61 ± 0.07 in female and 5.70 ± 0.09 in male, P > 0.05; Fig. 2B). The relaxing potency of raloxifene was greater in arteries constricted by 60 mM K⁺ than U46619 [GenBank] .

View larger version (19K): [in this window] [in a new window]   Fig. 2. Relaxation to raloxifene in (A) U46619 [GenBank] - or (B) 60 mM K+-constricted arteries with endothelium from female and male rats. Results are mean ± S.E.M. of seven or eight experiments.

Effect of Raloxifene on CaCl₂-Induced Constriction in Pulmonary Arteries. In Ca²⁺-free, 60 mM K⁺ solution, CaCl₂ induced constrictions of arteries without endothelium (pEC₅₀: 3.81 ± 0.15 in female and 3.77 ± 0.11 in male, P > 0.05). Raloxifene reduced CaCl₂-induced constrictions in a noncompetitive manner with progressive suppression of the maximal constriction in female (Fig. 3A) and male (Fig. 3B) arteries. In control experiments, 1 µM nifedipine abolished constrictions to CaCl₂.

View larger version (24K): [in this window] [in a new window]   Fig. 3. CaCl2-induced contraction in Ca2+-free, 60 mM K+ solution in the absence and presence of raloxifene (0.1-10 µM) in arteries without endothelium from (A) female and (B) male rats. Results are mean ± S.E.M. of six experiments.

Effects of Raloxifene on Pulmonary Veins. U46619 [GenBank] constricted pulmonary veins (tension: 0.96 ± 0.12 mN in female and 1.06 ± 0.23 mN in male, P > 0.05). In U46619 [GenBank] -constricted veins from both genders, raloxifene caused small relaxations, and this effect was similar in arteries with and without endothelium (Fig. 4, A and C). L-NAME did not modify this relaxation (Fig. 4, B and D). K⁺ (60 mM) constricted pulmonary veins (tension: 0.77 ± 0.12 mN in female and 0.87 ± 0.13 mN in male, P > 0.05), and this constriction was reduced by raloxifene (Fig. 5B). Gender-related differences were demonstrated in the venous responses to raloxifene, with a greater relaxing effect on male than female veins constricted by either U46619 [GenBank] or 60 mM K⁺ (Fig. 5, A and B).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effects of raloxifene on U46619 [GenBank] -constricted pulmonary veins from (A) female and (C) male rats. Lack of effect of 100 µM L-NAME on relaxation to raloxifene (B, female; D, male). Results are mean ± S.E.M. of seven or eight experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Gender differences in relaxant responses to raloxifene in pulmonary veins constricted by (A) U46619 [GenBank] or (B) 60 mM K+. Results are mean ± S.E.M. of seven or eight experiments. Statistical difference is indicated between two curves; ***, P < 0.001, two-way ANOVA.

Effect of Raloxifene on CaCl₂-Stimulated Increases in [Ca²⁺]_i in Pulmonary Arteries. The effect of raloxifene on [Ca²⁺]_i was examined in rings without endothelium. CaCl₂ induced [Ca²⁺]_i increase in Ca²⁺-free, 60 mM K⁺ solution, and the first and second concentration-dependent responses were similar. Changes in [Ca²⁺]_i measured as the fluorescence ratio (F340/F380) before and after treatment with raloxifene (0.3 and 10 µM) in female and male pulmonary arteries are summarized in Fig. 6. The cumulative addition of CaCl caused progressive increases in [Ca²⁺²]_i, and raloxifene reduced [Ca²⁺]_i increases. There was no gender difference in the effect of raloxifene (Fig. 6). In control experiments, 1 µM nifedipine abolished CaCl₂-induced increase in [Ca²⁺]_i.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Concentration-dependent increases in Ca2+/Fura-2 AM fluorescence in response to CaCl2 in a Ca2+-free, 60 mM K+-containing solution, and inhibitory effects of raloxifene (30-min incubation time) on rings from female (A and B) and male (C and D) rats. All the experiments were performed on pulmonary artery rings without endothelium that were first incubated with Fura-2 AM for 45 min and perfused with Ca2+-free, 60 mM K+ solution for 15 min before perfusion of 60 mM K+ solution supplemented with CaCl2 (0.1-3 mM). Changes in [Ca2+]i were recorded as the ratio (F340/F380) of Fura-2 fluorescence at excitation wavelengths of 340 and 380 nm. Data are expressed in mean ± S.E.M. of rings from four separate animals. Statistical difference is indicated between two curves; *, P < 0.05 and ***, P < 0.001, two-way ANOVA.

	Discussion

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The new findings from this study using isolated rat pulmonary arteries and veins are 1) raloxifene-induced pulmonary vascular relaxation was independent of the presence of endothelium; 2) raloxifene-induced acute effect is unrelated to ICI 182,780-sensitive estrogen receptors; 3) raloxifene reduced CaCl₂-induced constriction and Ca²⁺ influx through nifedipine-sensitive Ca²⁺ channels; and 4) there was a gender difference in vascular responses to raloxifene. Some of these observations parallel those made in previous studies on the systemic vessels (Figtree et al., 1999; Tsang et al., 2004b). This study of the acute effects of raloxifene suggests that raloxifene may reduce pulmonary pressure by inhibiting the activity of voltage-sensitive Ca²⁺ channels in vascular smooth muscle cells.

Raloxifene inhibited high K⁺-induced constrictions, indicating that raloxifene may act as a Ca²⁺ channel inhibitor to cause pulmonary vascular relaxation. Similar effects were reported in systemic arteries (Figtree et al., 1999; Tsang et al., 2004b). Raloxifene also reduced CaCl₂-induced constriction in high K⁺ solution with a progressive reduction of the maximal response; this further suggested that raloxifene interferes with Ca²⁺ influx through voltage-sensitive Ca²⁺ channels in these blood vessels. Ca²⁺ influx is the linker in excitation-contraction coupling in vascular smooth muscle on membrane depolarization or constrictor stimulation. The calcium antagonistic action of raloxifene was proven by the demonstration that raloxifene inhibits Ca²⁺ influx via Ca²⁺ channels, as revealed by [Ca²⁺]_i imaging measurement in Fura-2-loaded pulmonary vascular tissues without endothelium. The potency was similar for raloxifene between relaxing CaCl₂-induced tension and inhibiting CaCl₂-stimulated [Ca²⁺]_i increase. Like the L-type Ca²⁺ channel blocker nifedipine, raloxifene at 10 µM almost abolished CaCl₂-induced increase in vessel tone and [Ca²⁺]_i. High K⁺-constricted arteries also exhibited higher relaxing sensitivity to raloxifene than U46619 [GenBank] -constricted arteries. These data indicate that inhibition of Ca²⁺ entry via L-type Ca²⁺ channels is an important mechanism by which raloxifene causes pulmonary vascular relaxation.

Endothelial dysfunction characterized by progressive loss of the relaxation to NO-dependent dilators contributes to the development of hypoxic pulmonary hypertension (Adnot et al., 1991; Berkenbosch et al., 2000). Acute treatment with estrogen or phytoestrogens restored endothelial function in pulmonary arteries isolated from chronically hypoxic rats (Karamsetty et al., 2001). In the systemic circulation, raloxifene rapidly relaxed mammalian arteries and veins partly by increasing NO (Figtree et al., 1999; Bracamonte et al., 2002). However, the present study shows that raloxifene induced relaxation to the same extent in pulmonary arteries with and without endothelium. Inhibition of the NO pathway did not affect the relaxation. Despite the enhanced NO function described in the systemic arteries from raloxifene-treated rats (Wassmann et al., 2002), the present data make a positive role of endothelium/NO in the acute pulmonary relaxation to raloxifene unlikely. This conclusion agrees with the observation that estrogen attenuated pulmonary hypertension via an endothelial NO synthase-independent mechanism (Resta et al., 2001).

The contribution of estrogen receptors to vascular responses to estrogen or SERMs remains controversial and undefined. ICI 182,780, a selective estrogen receptor antagonist, inhibited the nongenomic effects of raloxifene on the endothelium (Figtree et al., 1999) but not on vascular smooth muscle (Figtree et al., 1999; Tsang et al., 2004b). The present study shows that ICI 182,780 failed to influence raloxifene-induced pulmonary artery relaxation. ICI 182,780 had no effect on relaxation to raloxifene in porcine femoral veins (Bracamonte et al., 2002). Instead, ICI 182,780 may act as a partial estrogen receptor agonist in femoral veins by causing relaxation (Bracamonte et al., 2002). However, ICI 182,780 did not induce significant relaxation in pulmonary arteries from both genders in the present study. Together, like its effects on some arteries in the systemic circulation (Bracamonte et al., 2002; Tsang et al., 2004b), the acute relaxation caused by raloxifene in pulmonary arteries in vitro does not involve ICI 182,780-sensitive estrogen receptor stimulation. However, it is unclear how raloxifene may act on Ca²⁺ channels in vascular smooth muscle if its effect is not mediated by estrogen receptors. Estrogen was shown to activate Ca²⁺-activated K⁺ channels by a direct interaction with the -subunit of the channel protein (Valverde et al., 1999). It is yet to elucidate whether the Ca²⁺ channel could provide such an interactive site for raloxifene. It should be noted that the present data do not preclude the chronic action of raloxifene on vascular estrogen receptors, which could contribute to long-term effects of raloxifene in the pulmonary circulation.

There is a gender difference in hypoxia-induced pulmonary hypertension (Rabinovitch et al., 1981) and right ventricular hypertrophy (McMurtry et al., 1973), but it is unknown whether this difference is influenced by the direct vascular effects of sex hormones. The gender difference was observed in relaxation to raloxifene of pulmonary arteries constricted by the receptor-dependent constrictor U46619 [GenBank] but not by the receptor-independent constrictor K⁺. The relaxing potency was higher in male than female arteries. This sexual dimorphism in raloxifene relaxation is more significant in pulmonary veins, regardless of the type of constrictors used. Finally, the present study shows that raloxifene was less effective in pulmonary veins than arteries, although the mechanism underlying that discrepancy is unclear.

In conclusion, the present findings provide experimental evidence for a key mechanism by which raloxifene relaxes rat pulmonary vessels. Raloxifene acts primarily on vascular smooth muscle of pulmonary arteries by inhibiting Ca²⁺ entry via L-type Ca²⁺ channels. This action is acute, nongenomic, and independent of a functional endothelium or ICI 182,780-sensitive estrogen receptors. Such calcium antagonistic action may make raloxifene a potentially useful agent in the pulmonary arterial hypertension like an oral Ca²⁺ channel blocker if this effect also occurs in vivo. Raloxifene is clinically used to treat menopausal women, but the present in vitro data show that raloxifene seems to be more effective in causing pulmonary vascular relaxation in male than female animals. However, it remains to be investigated whether raloxifene could exert similar gender-related effects in vivo on the pulmonary circulation.

	Footnotes

 
This study was supported by a grant from the Hong Kong Research Grants Council (CUHK 4366/03M), and F.P.L. is a postdoctoral fellow supported by this grant. Y.C.C. is the recipient of the Chinese University of Hong Kong Postgraduate Studentship.

doi:10.1124/jpet.104.077990.

ABBREVIATIONS: HRT, hormone replacement therapy; SERM, selective estrogen receptor modulator; NO, nitric oxide; U46619 [GenBank] , 9,11-dideoxy-11,9-epoxymethanoprostaglandin F₂; ICI 182,780, 7-[9-[(4,4,5,5,5,-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17-diol; AM, acetoxymethyl ester; L-NAME, N^G-nitro-L-arginine methyl ester; ODQ, 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one; ANOVA, analysis of variance.

Address correspondence to: Dr. Yu Huang, Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, Shatin, NT, Hong Kong. E-mail: yu-huang{at}cuhk.edu.hk

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